486
Biochimica et Biophysica Acta, 582 (1979) 486--495 © Elsevier/North-Holland Biomedical Press
BBA 28781 INHIBITION OF INSULIN RECEPTOR BINDING BY DIMETHYL SULFOXIDE
EMMANUEL VAN OBBERGHEN, PIERRE DE MEYTS * and JESSE ROTH Diabetes Branch, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda MD 20014 (U.S.A.)
(Received August 23rd, 1978) Key words: Insulin; Receptor binding; Dimethyl sulfoxide; (Negative cooperativity)
Summary Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured h u m a n lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of 12SI-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of 12sIlabeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor were affected to a much lesser extent.
Introduction The first step in the action of insulin is its binding to a specific receptor on the surface of the target cell [1]. The chemical nature of this interaction has been widely investigated by chemical modification of the insulin molecule [ 2 - 4 ] or by various enzymatic treatments directed at the receptor moiety * Present address: International Institute of Cellular and Molecular P a t h o l o g y , 7 5 a v e n u e H i p p o c r a t e , B - 1 2 0 0 Brussels, B e l g i u m . Abbreviations: Me2SO, d i m e t h y l s u l f o x i d e , H E P E S , 4 - ( 2 - h y d r o x y e t h y l ) - l - p i p e r a z i n e e t h a n e s u l f o n i c a c i d ; B/F, b o u n d / f r e e ; R 0 , t o t a l c o n c e n t r a t i o n o f r e c e p t o r sites p e r cell~ K , average affinity; Ke, limiting h i g h affinity state; Kf, limiting l o w a f f i n i t y s t a t e .
487 [5,6]. In contrast, little attention has been given to the possible role of interactions between the insulin or the receptor, on the one hand, and the milieu in which the insulin-receptor reaction takes place, on the other hand. This milieu is composed of two phases: the membrane and the solvent. In the fluid mosaic model of the cell membrane proposed by Singer and Nicolson [7] the membrane is pictured as a two-dimensional solution of integral proteins dispersed in a fluid lipid bilayer. The cell surface receptors for most hormones (e.g., insulin) are thought to be integral membrane proteins, associated to the membrane by strong hydrophobic bonds. Besides stabilizing hormone receptors in the membrane layer, hydrophobic bonding may play another important role in the function of polypeptide hormones. Indeed, Pullen et ai. [4] have suggested that hydrophobic forces, that is changes in the interaction of non-polar insulin residues with the water solvent, are crucial in the interaction of polypeptide hormones with their receptor, especially insulin and glucagon. Studies combining X-ray analysis, circular dichroism, and receptor binding of chemically modified insulins indicate that the three dimensional structure of insulin is of critical importance for the formation of the insulinreceptor complex and suggest that invariant nonpolar residues on the surface of the insulin monomer play a major role in receptor binding [4]. In a first attempt to the investigate the effect of the solvent on the reaction of insulin with its receptor, we have studied the effect of a polar solvent, dimethyl sulfoxide, on the binding of insulin to its cell surface receptors. Experimental procedures Materials Porcine insulin (7GU48L) was purchased from Elanco; Nal2SI {carrier free) from New England Nuclear; bovine serum albumin {Fraction V) from Miles Laboratories and dimethyl sulfoxide from Aldrich Chemical Co. All other chemicals were of reagent grade. Cultured human lymphocytes of the IM-9 cell line [8] were grown at 37°C in Eagle's minimum essential medium enriched with 10% fetal calf serum (International Biological Laboratories, Inc., RockviUe, Md.), 100 U/ml penicillin, 100 ~ig/ml streptomycin, and 0.29 mg/ml giutamine. Eagle's minimum essential medium was prepared in the Media Unit of the National Institutes of Health. The cells were 'fed' 3 times a week by dividing the cultures 1 : 3 and adding fresh media. Cells in log phase or in early stationary phase of growth were split 1 : 2 in fresh medium 24 h before being used. Methods 12SI-labeled insulin was prepared at specific activities of 150--200 Ci/g {approx. 0.5 atoms of iodine/insulin molecule) by a previously described modification of the chloramine-T method [9,10]. The methods used to study insulin binding to receptors on lymphocytes have been described in detail elsewhere [10] and are briefly summarized below. The assay buffer for these experiments was 100 mM HEPES, 120 mM NaCI, 1.2 mM MgSO4, 1 mM EDTA, 10 mM glucose, 15 mM sodium acetate, 10 mg/ml bovine serum albumin (pH 7.6). Labeled and unlabeled hormones as well as the cells were prepared in this buffer.
488 For the measurements of insulin binding to its receptor, 10 -1~ M '2SI-labeled insulin in the absence and presence of unlabeled hormone (1.67 • 10 -11 M-1.67 • 10 -6 M) was incubated with the lymphocytes (4 • 106 per assay in a final vol. of 0.5 ml) at 15°C for 90 min. Insulin binding was determined by layering 200-~1 aliquots onto 100 pl chilled assay buffer in 400 ~l plastic microfuge tubes. The microfuge tubes were then centrifuged, the supernatant was aspirated and the radioactivity in the cell pellet counted. Total binding refers to the radioactivity in the cell pellet, whereas the non-specific binding represents the radioactivity in the cell pellet in the presence of 1.67 • 10-6 M unlabeled insulin. Specific binding is the difference between total and non-specific binding. In some experiments, the ability of '2SI-labeled insulin to dissociate from the receptor was studied by adding to the incubation tube at steady-state (120 min) an excess of unlabeled insulin (1.67 • 10 -6 M) and monitoring the dissociation as a function of time. The negative cooperativity of the receptors was kinetically measured as previously described [ 11 ] by studying the dissociation of the insulin-receptor complex in an 'infinite' (100-fold) dilution, in the absence and in the presence of an excess of unlabeled insulin. For this study, cells at high concentration in a single batch were incubated for 90 min at 15°C with ~25I-labeled insulin at low concentration, such that only a small minority of the receptor sites were occupied by ~2SI-labeled insulin. At the end of the incubation, aliquots of 100 ~1 were immediately distributed to two sets of tubes at 15°C. The first set contained 10 ml hormone-free buffer ('dilution only') while the second set contained 10 ml buffer enriched with 1.67 • 10 -7 M unlabeled h o r m o n e ('dilution + unlabeled hormone'). The dissociation was monitored by centrifuging duplicate tubes from each set at regular intervals for 2 min at 700 × g. The supernatants were discarded and the radioactivity in the cell pellets was counted. D a t a analysis
The data on binding of 12SI-labeled insulin to receptor at steady state is plotted as bound/free ( B / F ) of ~2SI-labeled insulin as a function of bound hormone (Scatchard plot) [12]. For insulin binding to its receptors this plot is curvilinear. The total binding capacity or total concentration of receptor sites Ro is derived from the point where the linear extrapolation of the curve intersects the horizontal axis. Another m e t h o d of data analysis is the 'average affinity profile. Because the insulin receptor sites are not independent of one another, traditional methods for deriving the affinities of the receptors from curvilinear Scatchard plots are not valid [13]. Experimental data suggest that the insulin receptors are a single set of homogeneous binding sites that undergo negatively cooperative site-site interactions such that the affinity of the receptors for insulin is inversely related to the fractional occupancy [11,14]. The average affinity profile expresses the relationship between the average affinity for insulin (K) and the fraction of receptors occupied (Y) [15]. At any point i on the Scatchard curve, average affinity = K--i -
(B/F)i Ro - - B i
489 and fractional occupancy Y-~ = Bi/Ro, where B i = the concentration of bound hormone, ( B / F ) i = the bound/free hormone at that point, and R0 = total receptor concentration (for details of this analysis see [12]). When the log of the average affinity (K'-) is plotted as a function of the log of the fractional occupancy of the receptor (Y), the plot displays the average affinity of the receptor at all levels of receptor occupancy and is referred to as the 'average affinity profile' [15]. In this analysis, the limiting high affinity state, obtained at low levels of receptor occupancy, is designated Ke; the limiting low affinity state, obtained at high levels of receptor occupancy, is K-'-~.It should be noted that the validity of this analysis (and of the derived parameters) does not depend on assigning a particular model to the molecular mechanisms involved in the cooperativity [15]. Results
Inhibition of insulin binding by Me,SO Me2SO at concentrations exceeding 0.1% (v/v) produced a dose-related inhibition of 12SI-labeled insulin binding to IM-9 lymphocytes (Fig. 1). Me2SO induced also a dose-related increase in non-specifically bound 12SI-labeled insulin, so that in the presence of 20% Me2SO specific binding of insulin was nil. The effect of Me2SO on the binding of ~2SI-labeled insulin was immediate with no increase in effect upon prolonging (up to 3 h) the duration of exposure to the drug (data n o t shown). The inhibition of insulin binding observed in the presence of 1 and 10% Me2SO was almost totally reversed by washing the cells for 90 min in medium free of Me2SO (Table I, part A). Likewise, when 12sI100
z 8O-
•
.
P, ,z-tnO~
60
"-~
20 _ z~,, |
0.1%
NON-SPECIFIC BINDING ~, ~ , 1
I
t
'-T~
~
i
t It
1%
i
,~, I
l
I
I
I
I II
10%
I
I
I
i
i
i
i II
100%
Me2SO CONCENTRATION (V/V) Fig. 1. Effect of M e 2 S O o n insulin b i n d i n g t o IM-9 l y m p h o e y t e s . Cells (2.5 • 1 0 6 / m l ) weze p r e i n c u b a t e d in the absence a n d presence of Me2SO for 1 5 0 m l n a t 87~C f o l l o w e d b y 8 0 m i n a t 15°C. 1 2 5 I - l a b e l e d insulin ( 1 , 6 7 • 10 - I ! M), w i t h and w i t h o u t an e x c e s s o f u n l a b e l e d insulin ( 1 . 6 7 • 10 -~ M), w a s a d d e d and the i n c u b a t i o n c o n t i n u e d for a f ~ t h e r 9 0 r a i n a t 15°C. A t the e n d o f t h e i n c u b s t i o n , allquots w e r e eent1~f u g e d and the r a d i o a c t i v i t y in t h e cell pellet c o u n t e d for m e a s u r e m e n t s o f t o t a l b i n d i n g a n d non-specific b i n d i n g . T h e t o t a l and non-specific binding are p l o t t e d as a f u n c t i o n of t h e c o n c e n t r a t i o n of M e 2 S O . I n the absence of M e 2 S O the h o u n d / t o t a l 125 I-labeled insulin w a s 0 . 2 5 a n d w a s set as 1 0 0 % .
490 TABLE I R E V E R S I B I L I T Y O F T H E E F F E C T O F M e 2 S O O N T H E B I N D I N G O F I N S U L I N T O IM-9 L Y M P H O CYTES Part A: Cells (4 • 1 0 6 / m l ) w e r e p r e i n c u b a t e d f o r 1 5 0 rain a t 3 7 ° C w i t h o u t o r w i t h M e 2 S O (1 a n d 10%). A f t e r this p r e i n e u b a t i o n all cells w e r e e i t h e r w a s h e d w i t h assay b u f f e r f o r 9 0 rain at 3 7 ° C a n d s u b s e q u e n t ly s t u d i e d f o r 125 I-labeled insulin b i n d i n g , or s t u d i e d f o r 1 2 5 i . l a b e l e d insulin b i n d i n g i n a m e d i a t e l y a f t e r t h e p r e i n e u b a t i o n . F o r t h e d e t a i l e d d e s c r i p t i o n of t h e b i n d i n g assay see t h e l e g e n d t o Fig. 1. T h e b o u n d / free 1 2 5 i , l a b e l e d insulin o b s e r v e d w i t h t h e w a s h e d a n d n o n - w a s h e d cells p r e i n c u b a t e d in t h e a b s e n c e of M e 2 S O was set as 1 0 0 % . T h e b o u n d / f r e e 1 2 5 i . l a b e l e d insulin of t h e w a s h e d a n d n o n - w a s h e d p r e t r e a t e d w i t h Me 2SO was e x p r e s s e d as a p e r c e n t a g e o f t h e b o u n d / f r e e 1 2 5 i . l a b e l e d insulin of t h e c o r r e s p o n d i n g c o n t r o l cells. T h e n o n - s p e c i f i c b i n d i n g w h i c h has b e e n s u b t r a c t e d was less t h a n 4% of t h e t o t a l r a d i o a c t i v i t y in t h e a b s e n c e o r p r e s e n c e o f 1% M e 2 S O a n d e q u a l l e d 7% in t h e p r e s e n c e o f 10% M e 2 S O . Part B: 1 2 5 i . l a b e l e d insulin w a s i n c u b a t e d w i t h 10% a n d 1% M e 2 S O f o r 2.5 h a t 3 7 ° C a n d t h e n d i l u t e d to a final c o n c e n t r a t i o n o f 0.1% M e 2 S O . T h e b i n d i n g of t h e s e p r e t r e a t e d t r a c e r s was t h e n c o m p a r e d to t h e b i n d i n g of fresh 1 2 5 I - l a b e l e d insulin in t h e a b s e n c e or p r e s e n c e o f 1% M e 2 S O . F o r t h e details of t h e 1 2 5 i . l a b e l e d insulin b i n d i n g assay see l e g e n d to Fig. 1. T h e b o u n d / f r e e 125 I-labeled insulin o b s e r v e d w i t h t r a c e r , w h i c h has n o t seen Me 2 SO, w a s set as 1 0 0 % . T h u s , values q u o t e d are f o r 1 2 5 i . l a b e l e d insulin b o u n d , e x p r e s s e d as p e r c e n t a g e s o f c o n t r o l .
A Me 2 SO in b i n d i n g assay (%, v / v )
0 1 10
B Preincubation with r e c e p t o r (Me 2 SO concentration; %, v / v ) 1
10
99 81
89 -29
--
Me 2 SO in b i n d i n g assay (%, v / v )
0.1 1.0
Pteincubation with 125 I q a b e l e d insulin (Me 2 SO c o n c e n t r a t i o n ; %, v / v ) 1
10
100 76
86 --
labeled insulin tracer was pre-exposed to 10 or 1% Me, SO (v/v) and then after dilution to a final concentration of 0.1% (v/v) used in a binding assay, the effect of Me2SO was almost totally reversible (Table I, Part B).
Characteristics o f the Me2SO induced inhibition of insulin binding When cells were incubated with I or 10% Me,SO 12SI-labeled insulin binding, measured at steady state, was decreased at all concentrations of insulin (Fig. 2, left panel). The decrease in binding was more pronounced at low concentrations of insulin indicating a reduced affinity of the receptors (non-parallel displacem e n t of the Scatchard plot) w i t h o u t noticeable change in receptor concentration (horizontal intercept). Because insulin receptors show negative cooperativity in binding, the determination of receptor affinities were made by using the average affinity profile (Fig. 2, right panel). In the presence of 1 and 10% Me:SO the affinity at low levels of receptor occupancy (Ke) was reduced to 70% and 20% of the control values, respectively. Me2SO failed to significantly affect the affinity obtained at higher levels of receptor occupancy (Kf). The lack of effect of 0.1% Me2SO found in the Scatchard analysis was also reflected in the average affinity profile. The dissociation of ~2sI-tabeled insulin from its receptor and the phenomen o n of negative cooperativity were studied by the technique first described by De Meyts et al. [11]. Low concentrations of Me:SO (0.1 and 1%, v/v) failed to interfere with the dissociation of 125I-labeled insulin studied either by dilution only or dilution with unlabeled hormone (Fig. 3, left panel). The only effect on
491
AVERAGEAFFINITY"PROFILE
SCATCHARD ANALYSIS 0,8
CONTROL
MezSO0.1~--.-~ .o _z O.E
n,.
~
!~o
CONCENTRATION OF Me2SO(%, v/vl NONE o 0.1 •
0.4
10
oo~,
Me2SO 1% Z ,
Biochimica et Biophysica Acta, 582 (1979) 486--495 © Elsevier/North-Holland Biomedical Press
BBA 28781 INHIBITION OF INSULIN RECEPTOR BINDING BY DIMETHYL SULFOXIDE
EMMANUEL VAN OBBERGHEN, PIERRE DE MEYTS * and JESSE ROTH Diabetes Branch, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda MD 20014 (U.S.A.)
(Received August 23rd, 1978) Key words: Insulin; Receptor binding; Dimethyl sulfoxide; (Negative cooperativity)
Summary Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured h u m a n lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of 12SI-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of 12sIlabeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor were affected to a much lesser extent.
Introduction The first step in the action of insulin is its binding to a specific receptor on the surface of the target cell [1]. The chemical nature of this interaction has been widely investigated by chemical modification of the insulin molecule [ 2 - 4 ] or by various enzymatic treatments directed at the receptor moiety * Present address: International Institute of Cellular and Molecular P a t h o l o g y , 7 5 a v e n u e H i p p o c r a t e , B - 1 2 0 0 Brussels, B e l g i u m . Abbreviations: Me2SO, d i m e t h y l s u l f o x i d e , H E P E S , 4 - ( 2 - h y d r o x y e t h y l ) - l - p i p e r a z i n e e t h a n e s u l f o n i c a c i d ; B/F, b o u n d / f r e e ; R 0 , t o t a l c o n c e n t r a t i o n o f r e c e p t o r sites p e r cell~ K , average affinity; Ke, limiting h i g h affinity state; Kf, limiting l o w a f f i n i t y s t a t e .
487 [5,6]. In contrast, little attention has been given to the possible role of interactions between the insulin or the receptor, on the one hand, and the milieu in which the insulin-receptor reaction takes place, on the other hand. This milieu is composed of two phases: the membrane and the solvent. In the fluid mosaic model of the cell membrane proposed by Singer and Nicolson [7] the membrane is pictured as a two-dimensional solution of integral proteins dispersed in a fluid lipid bilayer. The cell surface receptors for most hormones (e.g., insulin) are thought to be integral membrane proteins, associated to the membrane by strong hydrophobic bonds. Besides stabilizing hormone receptors in the membrane layer, hydrophobic bonding may play another important role in the function of polypeptide hormones. Indeed, Pullen et ai. [4] have suggested that hydrophobic forces, that is changes in the interaction of non-polar insulin residues with the water solvent, are crucial in the interaction of polypeptide hormones with their receptor, especially insulin and glucagon. Studies combining X-ray analysis, circular dichroism, and receptor binding of chemically modified insulins indicate that the three dimensional structure of insulin is of critical importance for the formation of the insulinreceptor complex and suggest that invariant nonpolar residues on the surface of the insulin monomer play a major role in receptor binding [4]. In a first attempt to the investigate the effect of the solvent on the reaction of insulin with its receptor, we have studied the effect of a polar solvent, dimethyl sulfoxide, on the binding of insulin to its cell surface receptors. Experimental procedures Materials Porcine insulin (7GU48L) was purchased from Elanco; Nal2SI {carrier free) from New England Nuclear; bovine serum albumin {Fraction V) from Miles Laboratories and dimethyl sulfoxide from Aldrich Chemical Co. All other chemicals were of reagent grade. Cultured human lymphocytes of the IM-9 cell line [8] were grown at 37°C in Eagle's minimum essential medium enriched with 10% fetal calf serum (International Biological Laboratories, Inc., RockviUe, Md.), 100 U/ml penicillin, 100 ~ig/ml streptomycin, and 0.29 mg/ml giutamine. Eagle's minimum essential medium was prepared in the Media Unit of the National Institutes of Health. The cells were 'fed' 3 times a week by dividing the cultures 1 : 3 and adding fresh media. Cells in log phase or in early stationary phase of growth were split 1 : 2 in fresh medium 24 h before being used. Methods 12SI-labeled insulin was prepared at specific activities of 150--200 Ci/g {approx. 0.5 atoms of iodine/insulin molecule) by a previously described modification of the chloramine-T method [9,10]. The methods used to study insulin binding to receptors on lymphocytes have been described in detail elsewhere [10] and are briefly summarized below. The assay buffer for these experiments was 100 mM HEPES, 120 mM NaCI, 1.2 mM MgSO4, 1 mM EDTA, 10 mM glucose, 15 mM sodium acetate, 10 mg/ml bovine serum albumin (pH 7.6). Labeled and unlabeled hormones as well as the cells were prepared in this buffer.
488 For the measurements of insulin binding to its receptor, 10 -1~ M '2SI-labeled insulin in the absence and presence of unlabeled hormone (1.67 • 10 -11 M-1.67 • 10 -6 M) was incubated with the lymphocytes (4 • 106 per assay in a final vol. of 0.5 ml) at 15°C for 90 min. Insulin binding was determined by layering 200-~1 aliquots onto 100 pl chilled assay buffer in 400 ~l plastic microfuge tubes. The microfuge tubes were then centrifuged, the supernatant was aspirated and the radioactivity in the cell pellet counted. Total binding refers to the radioactivity in the cell pellet, whereas the non-specific binding represents the radioactivity in the cell pellet in the presence of 1.67 • 10-6 M unlabeled insulin. Specific binding is the difference between total and non-specific binding. In some experiments, the ability of '2SI-labeled insulin to dissociate from the receptor was studied by adding to the incubation tube at steady-state (120 min) an excess of unlabeled insulin (1.67 • 10 -6 M) and monitoring the dissociation as a function of time. The negative cooperativity of the receptors was kinetically measured as previously described [ 11 ] by studying the dissociation of the insulin-receptor complex in an 'infinite' (100-fold) dilution, in the absence and in the presence of an excess of unlabeled insulin. For this study, cells at high concentration in a single batch were incubated for 90 min at 15°C with ~25I-labeled insulin at low concentration, such that only a small minority of the receptor sites were occupied by ~2SI-labeled insulin. At the end of the incubation, aliquots of 100 ~1 were immediately distributed to two sets of tubes at 15°C. The first set contained 10 ml hormone-free buffer ('dilution only') while the second set contained 10 ml buffer enriched with 1.67 • 10 -7 M unlabeled h o r m o n e ('dilution + unlabeled hormone'). The dissociation was monitored by centrifuging duplicate tubes from each set at regular intervals for 2 min at 700 × g. The supernatants were discarded and the radioactivity in the cell pellets was counted. D a t a analysis
The data on binding of 12SI-labeled insulin to receptor at steady state is plotted as bound/free ( B / F ) of ~2SI-labeled insulin as a function of bound hormone (Scatchard plot) [12]. For insulin binding to its receptors this plot is curvilinear. The total binding capacity or total concentration of receptor sites Ro is derived from the point where the linear extrapolation of the curve intersects the horizontal axis. Another m e t h o d of data analysis is the 'average affinity profile. Because the insulin receptor sites are not independent of one another, traditional methods for deriving the affinities of the receptors from curvilinear Scatchard plots are not valid [13]. Experimental data suggest that the insulin receptors are a single set of homogeneous binding sites that undergo negatively cooperative site-site interactions such that the affinity of the receptors for insulin is inversely related to the fractional occupancy [11,14]. The average affinity profile expresses the relationship between the average affinity for insulin (K) and the fraction of receptors occupied (Y) [15]. At any point i on the Scatchard curve, average affinity = K--i -
(B/F)i Ro - - B i
489 and fractional occupancy Y-~ = Bi/Ro, where B i = the concentration of bound hormone, ( B / F ) i = the bound/free hormone at that point, and R0 = total receptor concentration (for details of this analysis see [12]). When the log of the average affinity (K'-) is plotted as a function of the log of the fractional occupancy of the receptor (Y), the plot displays the average affinity of the receptor at all levels of receptor occupancy and is referred to as the 'average affinity profile' [15]. In this analysis, the limiting high affinity state, obtained at low levels of receptor occupancy, is designated Ke; the limiting low affinity state, obtained at high levels of receptor occupancy, is K-'-~.It should be noted that the validity of this analysis (and of the derived parameters) does not depend on assigning a particular model to the molecular mechanisms involved in the cooperativity [15]. Results
Inhibition of insulin binding by Me,SO Me2SO at concentrations exceeding 0.1% (v/v) produced a dose-related inhibition of 12SI-labeled insulin binding to IM-9 lymphocytes (Fig. 1). Me2SO induced also a dose-related increase in non-specifically bound 12SI-labeled insulin, so that in the presence of 20% Me2SO specific binding of insulin was nil. The effect of Me2SO on the binding of ~2SI-labeled insulin was immediate with no increase in effect upon prolonging (up to 3 h) the duration of exposure to the drug (data n o t shown). The inhibition of insulin binding observed in the presence of 1 and 10% Me2SO was almost totally reversed by washing the cells for 90 min in medium free of Me2SO (Table I, part A). Likewise, when 12sI100
z 8O-
•
.
P, ,z-tnO~
60
"-~
20 _ z~,, |
0.1%
NON-SPECIFIC BINDING ~, ~ , 1
I
t
'-T~
~
i
t It
1%
i
,~, I
l
I
I
I
I II
10%
I
I
I
i
i
i
i II
100%
Me2SO CONCENTRATION (V/V) Fig. 1. Effect of M e 2 S O o n insulin b i n d i n g t o IM-9 l y m p h o e y t e s . Cells (2.5 • 1 0 6 / m l ) weze p r e i n c u b a t e d in the absence a n d presence of Me2SO for 1 5 0 m l n a t 87~C f o l l o w e d b y 8 0 m i n a t 15°C. 1 2 5 I - l a b e l e d insulin ( 1 , 6 7 • 10 - I ! M), w i t h and w i t h o u t an e x c e s s o f u n l a b e l e d insulin ( 1 . 6 7 • 10 -~ M), w a s a d d e d and the i n c u b a t i o n c o n t i n u e d for a f ~ t h e r 9 0 r a i n a t 15°C. A t the e n d o f t h e i n c u b s t i o n , allquots w e r e eent1~f u g e d and the r a d i o a c t i v i t y in t h e cell pellet c o u n t e d for m e a s u r e m e n t s o f t o t a l b i n d i n g a n d non-specific b i n d i n g . T h e t o t a l and non-specific binding are p l o t t e d as a f u n c t i o n of t h e c o n c e n t r a t i o n of M e 2 S O . I n the absence of M e 2 S O the h o u n d / t o t a l 125 I-labeled insulin w a s 0 . 2 5 a n d w a s set as 1 0 0 % .
490 TABLE I R E V E R S I B I L I T Y O F T H E E F F E C T O F M e 2 S O O N T H E B I N D I N G O F I N S U L I N T O IM-9 L Y M P H O CYTES Part A: Cells (4 • 1 0 6 / m l ) w e r e p r e i n c u b a t e d f o r 1 5 0 rain a t 3 7 ° C w i t h o u t o r w i t h M e 2 S O (1 a n d 10%). A f t e r this p r e i n e u b a t i o n all cells w e r e e i t h e r w a s h e d w i t h assay b u f f e r f o r 9 0 rain at 3 7 ° C a n d s u b s e q u e n t ly s t u d i e d f o r 125 I-labeled insulin b i n d i n g , or s t u d i e d f o r 1 2 5 i . l a b e l e d insulin b i n d i n g i n a m e d i a t e l y a f t e r t h e p r e i n e u b a t i o n . F o r t h e d e t a i l e d d e s c r i p t i o n of t h e b i n d i n g assay see t h e l e g e n d t o Fig. 1. T h e b o u n d / free 1 2 5 i , l a b e l e d insulin o b s e r v e d w i t h t h e w a s h e d a n d n o n - w a s h e d cells p r e i n c u b a t e d in t h e a b s e n c e of M e 2 S O was set as 1 0 0 % . T h e b o u n d / f r e e 1 2 5 i . l a b e l e d insulin of t h e w a s h e d a n d n o n - w a s h e d p r e t r e a t e d w i t h Me 2SO was e x p r e s s e d as a p e r c e n t a g e o f t h e b o u n d / f r e e 1 2 5 i . l a b e l e d insulin of t h e c o r r e s p o n d i n g c o n t r o l cells. T h e n o n - s p e c i f i c b i n d i n g w h i c h has b e e n s u b t r a c t e d was less t h a n 4% of t h e t o t a l r a d i o a c t i v i t y in t h e a b s e n c e o r p r e s e n c e o f 1% M e 2 S O a n d e q u a l l e d 7% in t h e p r e s e n c e o f 10% M e 2 S O . Part B: 1 2 5 i . l a b e l e d insulin w a s i n c u b a t e d w i t h 10% a n d 1% M e 2 S O f o r 2.5 h a t 3 7 ° C a n d t h e n d i l u t e d to a final c o n c e n t r a t i o n o f 0.1% M e 2 S O . T h e b i n d i n g of t h e s e p r e t r e a t e d t r a c e r s was t h e n c o m p a r e d to t h e b i n d i n g of fresh 1 2 5 I - l a b e l e d insulin in t h e a b s e n c e or p r e s e n c e o f 1% M e 2 S O . F o r t h e details of t h e 1 2 5 i . l a b e l e d insulin b i n d i n g assay see l e g e n d to Fig. 1. T h e b o u n d / f r e e 125 I-labeled insulin o b s e r v e d w i t h t r a c e r , w h i c h has n o t seen Me 2 SO, w a s set as 1 0 0 % . T h u s , values q u o t e d are f o r 1 2 5 i . l a b e l e d insulin b o u n d , e x p r e s s e d as p e r c e n t a g e s o f c o n t r o l .
A Me 2 SO in b i n d i n g assay (%, v / v )
0 1 10
B Preincubation with r e c e p t o r (Me 2 SO concentration; %, v / v ) 1
10
99 81
89 -29
--
Me 2 SO in b i n d i n g assay (%, v / v )
0.1 1.0
Pteincubation with 125 I q a b e l e d insulin (Me 2 SO c o n c e n t r a t i o n ; %, v / v ) 1
10
100 76
86 --
labeled insulin tracer was pre-exposed to 10 or 1% Me, SO (v/v) and then after dilution to a final concentration of 0.1% (v/v) used in a binding assay, the effect of Me2SO was almost totally reversible (Table I, Part B).
Characteristics o f the Me2SO induced inhibition of insulin binding When cells were incubated with I or 10% Me,SO 12SI-labeled insulin binding, measured at steady state, was decreased at all concentrations of insulin (Fig. 2, left panel). The decrease in binding was more pronounced at low concentrations of insulin indicating a reduced affinity of the receptors (non-parallel displacem e n t of the Scatchard plot) w i t h o u t noticeable change in receptor concentration (horizontal intercept). Because insulin receptors show negative cooperativity in binding, the determination of receptor affinities were made by using the average affinity profile (Fig. 2, right panel). In the presence of 1 and 10% Me:SO the affinity at low levels of receptor occupancy (Ke) was reduced to 70% and 20% of the control values, respectively. Me2SO failed to significantly affect the affinity obtained at higher levels of receptor occupancy (Kf). The lack of effect of 0.1% Me2SO found in the Scatchard analysis was also reflected in the average affinity profile. The dissociation of ~2sI-tabeled insulin from its receptor and the phenomen o n of negative cooperativity were studied by the technique first described by De Meyts et al. [11]. Low concentrations of Me:SO (0.1 and 1%, v/v) failed to interfere with the dissociation of 125I-labeled insulin studied either by dilution only or dilution with unlabeled hormone (Fig. 3, left panel). The only effect on
491
AVERAGEAFFINITY"PROFILE
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